1. Field of the Invention
The present invention relates to a medical diagnostic for investigating tissue components. More particularly, the present invention relates to a micro-scale instrument that utilizes autofluorescence emission and/or polarized elastic light scattering for real-time monitoring of microstructures and cells in tissues.
2. Description of Related Art
The diagnostic gold-standard of histological evaluation of living tissues typically entails fixation, sectioning, and staining to obtain thin samples which exhibit high contrast under the microscope. While this process has led to a much deeper understanding of cellular structure, tissue sectioning is time consuming, requires removal of tissue from the patient, and has inherent sampling error. However, the most important limitation is the delay, due to processing, in providing the surgeon with clinically relevant information at the time of surgery. While frozen section analysis is an accepted practice, this technique can be used only for readily identifiable lesions. Thus, there is clearly a need to develop new methods to complement existing modalities by providing the surgeon real time information that could be used intra-operatively to identify suspect lesions.
In recent years, technological developments in laser and detection instrumentation have facilitated the exploration of optical spectroscopic techniques for the detection and monitoring of disease at the tissue level. A number of spectroscopic approaches utilizing tissue autofluorescence and/or light scattering have led the way in the development of photonic methods for in-vivo characterization of tissue structures. Background information for such techniques can be found in: “Laser induced fluorescence spectroscopy from native cancerous and normal tissues”, by R. R. Alfano, B. Tata, J. Cordero, P. Tomashefsky, F. W. Longo. M. A. Alfano IEEE J. Quantum Electron., 20, 1507-1511 (1984); “Laser-induced fluorescence spectroscopy of human colonic mucosa detection of adenomatous transformation”, by C. R. Kapadia, F. W. Cutruzzola, K. M. O'Brien, M. L. Stetz, R. Enriquez, L. I. Deckelbaum, Gastroenterology, 99, 150-157 (1990); “Spectroscopic diagnosis of colonic dysphasia”, by R. R. Kortum, R. P. Rava, R. E. Petras, M. Fitzmaurice, M. Sivak, M. S. Feld, Photochem & Photobio., 53, 777-786, (1991); “Characterization of human breast specimens with Near-IR Raman spectroscopy”, by C. J. Frank, D. C. Redd, T. S. Gansler, R. L. McCreery, Anal. Chem., 66, 319-326 (1994); “Cervical precancer detection using a multivariate statistical algorithm based on laser-induced fluorescence spectra at multiple excitation wavelengths”, by N. Ramanujam, M. F. Mitchell, A. MahadevanJansen, S. L. Thomsen, G. Staerkel, A. Malpica, T. Wright, N. Atkinson, R. Richards-Kortum, Photochemistry and Photobiology, 64, 720-735(1996); and “Detection of preinvasive cancer cells”, by V. Backman, M. B. Wallace, L. T. Perelman, J. T. Arendt, R. Gurjar, M. G. Muller, Q. Zhang, G. Zonios, E. Kline, T. McGillican, S. Shapshay, T. Valdez, K. Badizadegan, J. M. Crawford, M. Fitzmaurice, S. Kabani, H. S. Levin, M. Seiler, R. R. Dasari, I. Itzkan, J. Van Dam, M. S. Feld, Nature, 406, 35-36 (2000).
Although these techniques have been explored extensively at the macroscopic level for more than a decade, their recent adaptation to the microscopic level has demonstrated their capability to image tissue micro-structures directly correlated to the histopathology of the tissue. Using confocal microscopy, tissue imaging at the microscopic level has been demonstrated using autofluorescence and light scattering. Background information on such methods and instrumentation can be found in: “Quantitative laser scanning confocal autofluorescence microscopy of normal, premalignant, and malignant colonic tissues”, by W. Hsing-Wen, J. Willis, M. J. F. Canto, M. V. Jr. Sivak, J. A. Izatt, IEEE Transactions on Biom. Engineering, 46, 101246-52 (1999); “Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy”, by I. Pavlova, K. Sokolov, R. Drezek, A. Malpica, M. Follen, R. Richards-Kortum, Photochem Photobiol., 77, 550-555 (2003); “Laser-induced autofluorescence microscopy of normal and tumor human colonic tissue”, by Z. W. Huang, W. Zheng, S. S. Xie, R. Chen, H. S. Zeng, D. I. McLean, H. Lui, Int. J. l Of Oncology, 24, 59-63 (2004); “In vivo, real-time confocal imaging”, by J. V. Jester, P. M. Andrews, W. M. Petroll, M. A. Lemp, H. D. Cavanagh, J Electron Microsc Tech. 18, 50-60, (1991), and “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology”, by M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, S. Gonzalez., J. of Investigative Dermatology, 117, 1137-1143 (2001).
The development of ultrafast lasers stimulated the utilization of nonlinear interactions of ultrashort pulses with cell components. Second harmonic generation imaging arises only by molecules which are noncentrosymmetric, and hence contrast is a function of the molecular structure of the specimen and its orientation with respect to the laser beam. Two-photon laser scanning microscopy offers higher resolution than confocal microscopy using infrared pulses for excitation. Coherent anti-Stokes Raman scattering microscopy offers the possibility for imaging by targeting specific molecular species. Background information on such nonlinear imaging techniques can be found in: “Second harmonic imaging in the scanning optical microscope” by J. N. Gannaway, C. J. R. Sheppard, Optical and Quantum Electronics, 10, 435-439 (1978); “Two-photon Laser scanning fluorescence microscopy” by W. Denk, J. H. Strickler, W. W. Webb, Science, 248, 73-76 (1990); and “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering” by A. Zumbusch, G. R. Holtom, and X. S. Xie, Phys. Rev. Lett., 82, 4142-4145 (1999).
These nonlinear imaging techniques offer unique diagnostic capabilities, however their implementation in a clinical environment requires the accommodation of complex instrumentation and specialized technical expertise. On the other hand, confocal microscopy is less complex but the light collection efficiency is only a small fraction of that of conventional fluorescence microscopy. In addition, the focused beam used in confocal microscopy leads to even less efficient autofluorescence signal collection due to photo-bleaching of native tissue fluorophores and sets a limitation on the excitation energy permissible thus increasing the necessary integration times. Furthermore, it is very difficult to incorporate hyperspectral imaging techniques in a confocal microscope without major compromises in the instrument's size and cost. Although these issues may be easily resolvable for some applications, the in-vivo application of these advanced microscopies in a clinical setting may be proven challenging.
Accordingly, a need exists for a microscopy system and method that incorporates hyperspectral/multimodal imaging while offering high spatial resolution and optimized signal sensitivity for fast image acquisition. The present invention is directed to such a need.